Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor

Ivan, Mircea; Haberberger, Thomas; Gervasi, David C.; Michelson, Kristen; Günzler, Volkmar; Kondo, Keiichi; Yang, Haifeng; Sorokina, Irina; Conaway, Ronald C.; Conaway, Joan Weliky; Kaelin, William G.

doi:10.1073/pnas.192342099

Cited by 524 publications

(406 citation statements)

References 40 publications

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“…26 Indeed, the hydroxylation of the HIFa protein can be inhibited by iron chelators or with dimethyloxallyl glycine, a competitive inhibitor with respect to 2-oxoglutarate. 27 The hydroxylated proline residues of the HIFa protein are then recognized by an E3 ubiquitin ligase, von Hippel-Lindau protein (pVHL), which subsequently targets HIF for rapid degradation by the proteasome. [28][29][30] An inherited deficiency in pVHL allows HIF to become constitutively active, leading to renal cell carcinoma, further illustrating the importance of HIF in cancer pathology.…”

Section: Regulation Of Hif Functionmentioning

confidence: 99%

Mitochondrial complex III regulates hypoxic activation of HIF

2008

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Decreases in oxygen levels are observed in physiological processes, such as development, and pathological situations, such as tumorigenesis and ischemia. In the complete absence of oxygen (anoxia), mammalian cells are unable to generate sufficient energy for survival, so a mechanism for sensing a decrease in the oxygen level (hypoxia) before it reaches a critical point is crucial for the survival of the organism. In response to decreased oxygen levels, cells activate the transcription factors hypoxiainducible factors (HIFs), which lead to metabolic adaptation to hypoxia, as well as to generate new vasculature to increase oxygen supply. How cells sense decreases in oxygen levels to regulate HIF activation has been hotly debated. Emerging evidence indicates that reactive oxygen species (ROS) generated by mitochondrial complex III are required for hypoxic activation of HIF. This review examines the current knowledge about the role of mitochondrial ROS in HIF activation, as well as implications of ROS-level regulation in pathological processes such as cancer. Oxygen Homeostasis and HIF Maintaining oxygen homeostasis is critical for survival and proper function of cells and organisms. 1 Reduced oxygen levels (hypoxia) initiates proper placental and vascular development. Hypoxia also has a causal role in pathological conditions such as ischemia-related diseases and cancer. A complete absence of oxygen (anoxia) results in cell death. 2 ATP synthesis is ablated, and most cells undergo apoptosis soon after anoxia exposure. 3-5 Cells exposed to hypoxia, or reduced oxygen levels, on the other hand, are able to maintain normal ATP synthesis and survive. 5,6 However, as cells replicate in a hypoxic environment, they reduce the oxygen supply even further and generate levels of local anoxia. Therefore, cells must respond quickly to decreasing oxygen levels before reaching an anoxic state (Figure 1).Mammalian cells respond to hypoxia by activating broadaction transcription factors named hypoxia-inducible factors, or HIFs, which are expressed by virtually all cells of the body. 7-9 Three members of the HIF family exist, named HIF1, HIF2 and HIF-3. 10-13 HIFs bind to hypoxia-responsive elements, consensus sequences in the promoter region of more than one hundred genes, activating the transcription of genes that allow the cell to adapt to and survive in the hypoxic environment. 14,15 Genes regulated by HIFs include glucose transporter that allow the cells to efficiently import glucose to continue generating ATP despite reduced nutrient availability; and genes that reorganize the microenvironment to bring in oxygen, such as vascular endothelial growth factor, which stimulates formation of new blood vessels. [16][17][18] Importantly for tumorigenesis, HIF also turns on genes such as insulin-like growth factor 2,19 which induces cellular survival and proliferation, as well as those that promote tumor invasion and migration, such as matrix metalloproteinase-2. 20 These factors contribute to tumor growth and invasion and metastasis, im...

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Section: Regulation Of Hif Functionmentioning

confidence: 99%

Mitochondrial complex III regulates hypoxic activation of HIF

2008

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“…The HIF prolyl-4-hydroxylase domain proteins, PHD1, 2 and 3 are responsible for normoxic HIF-a proteolysis. Prolyl-4-hydroxylation is necessary for the interaction with the von Hippel-Lindau tumor suppressor protein (pVHL) that mediates HIF-a degradation (Maxwell et al, 1999;Bruick and McKnight, 2001;Epstein et al, 2001;Jaakkola et al, 2001;Ivan et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Prolyl-4-hydroxylase PHD2- and hypoxia-inducible factor 2-dependent regulation of amphiregulin contributes to breast tumorigenesis

et al. 2010

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Hypoxia-elicited adaptations of tumor cells are essential for tumor growth and cancer progression. Although ample evidence exists for a positive correlation between hypoxiainducible factors (HIFs) and tumor formation, metastasis and bad prognosis, the function of the HIF-a protein stability regulating prolyl-4-hydroxylase domain enzyme PHD2 in carcinogenesis is less well understood. In this study, we demonstrate that downregulation of PHD2 leads to increased tumor growth in a hormone-dependent mammary carcinoma mouse model. Tissue microarray analysis of PHD2 protein expression in 281 clinical samples of human breast cancer showed significantly shorter survival times of patients with low-level PHD2 tumors over a period of 10 years. An angiogenesis-related antibody array identified, amongst others, amphiregulin to be increased in the absence of PHD2 and normalized after PHD2 reconstitution. Cultivation of endothelial cells in conditioned media derived from PHD2-downregulated cells resulted in enhanced tube formation that was blocked by the addition of neutralizing anti-amphiregulin antibodies. Functionally, amphiregulin was regulated on the transcriptional level specifically by HIF-2 but not HIF-1. Our data suggest that PHD2/HIF-2/amphiregulin signaling has a critical role in the regulation of breast tumor progression and propose PHD2 as a potential tumor suppressor in breast cancer.

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“…On the molecular level, cells sense alterations in oxygen levels by oxygen-dependent HIF prolyl and asparaginyl hydroxylation, which determines HIF-a protein stability and transactivation activity, respectively. Oxygen-dependent hydroxylation of two distinct HIF-a prolyl residues by the three HIF prolyl-4-hydroxylase domain proteins, PHD1, 2 and 3 (alternatively termed HPH3/EGLN2, HPH2/EGLN1 and HPH1/EGLN3, respectively), is necessary for the interaction with the von Hippel-Lindau tumor suppressor protein (pVHL) that serves as a recognition unit of a multiprotein ubiquitin E3 ligase and targets HIF-a for proteasomal degradation (Maxwell et al, 1999;Bruick and McKnight, 2001;Epstein et al, 2001;Jaakkola et al, 2001;Ivan et al, 2002). Compared with collagen prolyl-4-hydroxylase and mitochondrial cytochrome c oxidase, PHDs have a lower O 2 affinity that suits these enzymes to regulate HIF-a protein levels in response to a wide range of physiologically relevant pO 2 (Schofield and Ratcliffe, 2004).…”

Section: Introductionmentioning

confidence: 99%

Onconeuronal cerebellar degeneration-related antigen, Cdr2, is strongly expressed in papillary renal cell carcinoma and leads to attenuated hypoxic response

Balamurugan

et al. 2009

Oncogene

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The onconeuronal cerebellar degeneration-related antigen Cdr2 is associated with paraneoplastic syndromes. Neoplastic expression of Cdr2 in ovary and breast tumors triggers an autoimmune response that suppresses tumor growth by developing tumor immunity, but culminates in cerebellar degeneration when Cdr2-specific immune cells recognize neuronal Cdr2. We identified Cdr2 as a novel interactor of the hypoxia-inducible factor (HIF) prolyl-4-hydroxylase PHD1 and provide evidence that Cdr2 might represent a novel important tumor antigen in renal cancer. Strong Cdr2 protein expression was observed in 54.2% of papillary renal cell carcinoma (pRCC) compared with 7.8% of clear-cell RCC and no staining was observed in chromophobe RCC or oncocytoma. High Cdr2 protein levels correlated with attenuated HIF target gene expression in these solid tumors, and Cdr2 overexpression in tumor cell lines reduced HIF-dependent transcriptional regulation. This effect was because of both attenuation of hypoxic protein accumulation and suppression of the transactivation activity of HIF-1a. pRCC is known for its tendency to avascularity, usually associated with a lower pathological stage and higher survival rates. We provide evidence that Cdr2 protein strongly accumulates in pRCC, attenuates the HIF response to tumor hypoxia and may become of diagnostic importance as novel renal tumor marker.

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Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor

Cited by 524 publications

References 40 publications

Mitochondrial complex III regulates hypoxic activation of HIF

Mitochondrial complex III regulates hypoxic activation of HIF

Prolyl-4-hydroxylase PHD2- and hypoxia-inducible factor 2-dependent regulation of amphiregulin contributes to breast tumorigenesis

Onconeuronal cerebellar degeneration-related antigen, Cdr2, is strongly expressed in papillary renal cell carcinoma and leads to attenuated hypoxic response

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